The DNA damage Response (DDR) is a regulatory network that coordinates cellular processed in response to DNA damage and DNA replication stress. In addition, it directly orchestrates DNA repair choices because senses different DNA damage structures and transduces that information in cis to activate specific an appropriate repair options, thereby optimizing repair. The importance of the DDR is highlighted by the numerous cancer redisposition syndromes that result from its inactivation, including hereditary breast cancer resulting from BRCA1/2 mutation, AT, XP, and numerous others. Furthermore, targeted inhibition of the DDR can be exploited to enhance tumor sensitivity to radiation and genotoxic chemotherapy as well a synthetic lethality with the successful treatment of BRCA1/2-deficient tumors with PARP inhibitors. In the last 20 years we and others have investigated the composition of the central sensing and signaling apparatus used to detect and respond to genotoxic stress, first in yeast and more recently in mammals. This has revealed a conserved core of sensing and signaling proteins between yeast and mammals, just as there has been for classical DNA repair pathways. However, our analysis of substrates of this DDR kinase cascade has revealed an extremely diverse set of proteins and functions contacted by the DDR in mammals and the great majority of these activities are not conserved in yeast. Our analysis of ATM and ATR substrates and our and other analyses of non-ATM/ATR regulated phosphorylation events have implicated over 1000 proteins in the DDR the vast majority of which have no previous links to the DDR and have no yeast counterparts. Therefor there are likely to be many new components of the DDR to be discovered in mammals and it is critical that we set out to identify these factors in order to generate a complete understanding of the DDR and its significance in cellular and organismal physiology. Toward this end, we have developed sophisticated genetic tools that allow us to employ RNAi to find new protein candidates involved in promoting survival in response to DN damage. We have performed three preliminary screens to find new DDR candidate proteins that will serve at the basis for AIM1 and AIM2. In these AIMs we propose to carry out exhaustive validation of the candidate to identify bona fide new DDR proteins and will follow up on a candidate protein already validated. I addition we have also developed cutting edge proteomics methods to quantitatively detect post-translation modifications in response to DNA damage that form the basis of AIM3. In this AIMs we propose to identify new DDR proteins that are modified by ubiquitin and will follow one such protein that is known to regulate DNA damage foci formation. We also propose to use new proteomic and in situ localization screening to identify new proteins that localize to sites of DNA damage in AIM4. These innovative methods will allow us to deeply probe the layers of the DDR to identify and prioritize new proteins and to then probe the function of these proteins in the DDR using approaches we have pioneered for so many years.